The health of the animals is the main key to obtain healthy and quality food. Thus, the control of the diseases and the use of all the available tools thereof is of outmost importance. Vaccination is one more of all the tools used daily in the farms, existing other tools as biosecurity measures so as to avoid the entrance of pathogens coming from other exploitations, the application of hygiene standards and the management of animals for reducing the dissemination of diseases among the animals of the same exploitation, strict feeding control, or the creation of a comfortable environment for the animals of the farm.
Notwithstanding, to date there are diseases which are not appropriately controlled. Among these diseases we find those produced by viruses, such as the one caused by the porcine reproductive and respiratory syndrome, which is a severe disease in the porcins, and which was reported in the United States in 1987 and later identified in many other European countries. In 1991, the isolation of the etiological agent was reported in Holland and was named as Lelystad virus, and due to the symptomatology observed in the pigs it was known as porcine respiratory and/or epidemic abortion.
Another viral disease which has not been controlled is the Porcine Epidemic Diarrhea (PED), which is a viral disease exclusive of the porcins, very contagious and in most of the cases it leads to death. This disease affects the digestive system and the suckling pigs die in a term of 3-5 days due to diarrhea and dehydration.
Another disease is the one caused by the white spot syndrome virus (WSSV), the main pathogen of the shrimp and responsible of great production and incomings losses in the farm industry worldwide. Up to day, there is no effective treatment for controlling the infection.
The treatment of the diseases caused by bovine coronavirus and rotavirus is also relevant.
Moreover, we find the diseases caused by bacteria, such as the bovine mastitis which produces inflammation of the mammary gland and its secreting tissues, thereby reducing the production of the milk volume and altering its composition, and even its flavor, besides increasing its normal bacterial load. According to its duration, it may be classified in acute or chronic disease. Regarding its clinical expression, it may be clinical or subclinical. This disease causes severe economic losses to the dairy industry.
We also find the diseases caused by Actinobacillus pleuropneumoniae, the bacteria responsible for respiratory disorders in porcins with worldwide distribution, being known 50 years ago, and having and increased occurrence since the 1980 decade, being frequent in feedlots. It is the main responsible for the porcine pleuropneumonia, as well as for the agent which is directly involved in the Porcine Respiratory Complex. This is a high-dissemination disease, highly contagious and in many cases lethal for porcins from the weaning to the sacrifice. It causes fibrinous pleuritis with very characteristic costal adherence in 30-50% of the porcins, and an increased mortality in acute events, with increased growing delay in the chronic events. Furthermore, it has been found that the Actinobacillus pleuropneumoniae is involved in the cases of otitis media, arthritis and osteomyelitis.
The treatment of the diseases caused by E. coli, Salmonella spp and Clostridium perfringens is also relevant for the health of the animals.
Furthermore, there is coccidiosis which is an infectious disease caused by strict intracellular life parasites of the Eimeria spp. and Isospora spp. genus. Coccidias are omnipresent as they exist in most of the cattle facilities worldwide. These parasites may infect a wide variety of animals including humans, poultry, ruminants, pigs, dogs, cats and other domestic animals, nevertheless in most of the cases, coccidias are specific species.
Additionally, other health problems faced by the cattle industry are those caused by trichothecenes which are toxins produced by several Fusarium fungi, particularly Fusarium graminearum and Fusarium sporotrichioides. They are produced in crops and they enter into the food through polluted ingredients. Trichothecenes are proven tissue irritants and its intake is mainly associated with oral injuries, dermatitis and intestinal irritation. The main physiological response to these mycotoxins is the loss of appetite. Thrichothecenes are strong suppressing mycotoxins affecting the immune cellular response with a direct impact over the marrow, spleen, lymphoid tissues, thymus and intestinal mucosa, where the actively divided cells are injured.
For the preventive control of all the above diseases, there are basically two forms of protection. They may be exposed to infectious agent-derived antigens for the stimulation of a protective immune reaction, or they may be administered with a preformed antibody obtained from an immune subject.
The first form of protection is achieved by vaccines which may be of different classes: live microorganisms, lyophilized, or dead in oily emulsions, and recently, the creation of cloned and recombinant vaccines. Each of them has advantages and disadvantages regarding protection, immune response and lasting of the protection. Nevertheless, it has been found that in some cases, there are undesired injuries in the host due to the vaccine virus (Tizard, I. R. 1998. Vacunación y vacunas In: Inmunologia Veterinaria. 5th. Edition, Mc. Graw-Hill. Pp. 285-305).
The second form of protection is also called passive immunity and involves the transmission of specific antibodies against infectious agents to a host.
Traditionally, at the research level, the antibodies are made mainly in mammals and less frequently in poultries. The types of antibodies which are regularly made in mammals are monoclonal and polyclonal, and polyclonal in poultries (Larsson, et al., 1993. Chicken antibodies: taking advantage of evolution. A review Poultry Sci. 72: 1807-1812).
In the case of poultries, the Gailus gallus domesticus species (roosters and hens) is the only species from which the antibodies are obtained in a more accessible way and in a highly defined manner. The main serum antibody that is present in said species is lgG, although lgG is carried to the egg in a similar way to the transference of the mammal lgG through the placenta.
In the egg, immunoglobulin Y (IgY) is also present in a higher concentration in the egg yolk, nevertheless, there are also small amounts of IgY in the egg white. There has even been found that the amounts of IgY are higher in the egg yolk than in the hen serum (Larsson, et al 1993. Chicken antibodies: taking advantage of evolution. A review Poultry Sc. 72: 1807-1212).
In order to have an idea of the amount of antibodies produced by hens, it will suffice to note that a laying hen produces approximately 5 to 6 eggs per week with an approximate egg yolk volume of 15 ml, thus, within a week, a hen may produce egg yolk antibodies equivalent to 90 to 100 ml of serum or 180 to 200 of whole blood. This, when compared against the 20 ml of whole blood given by an immunized rabbit per week, allows us to clearly note the efficient productivity of the antibodies in egg yolk. Obviously, if bigger animals are used, such as horses or cows, the amount of serum and antibodies would be higher than in the egg, but this procedure is expensive and also more painful for the animals.
Among the advantages of the egg yolk antibodies, there are:
1. They do not bind the complement.
2. They do not bind to Staphilococcus aureus A Protein.
3. They do not react to the Rheumatoid factor.
4. Due to their philogenetics difference with mammal antibodies, IgY does not show crossed reaction with mammal antibodies.
5. Low production cost.
In the recent years, the egg yolk antibodies (immunoglobulins) have been used as diagnosis and therapy tools (Schmidt, et al. 1989). Thus, taking advantage of their philogenetics difference with mammal antibodies, Ig's have shown several advantages when used in immunodiagnosis. For example, egg yolk Ig's have been used for detecting several viruses by means of ELISA, immunodiffusion, immunofluorescence and complement fixation techniques. Due to their low isoelectric point as compared with the humans Ig, it has been used in electrophoresis assays for quantifying immunoglobulins in several animals serum (Altschuh, D. et al. 1984. Determination of IgG and IgM levels in serum by Rocket Immunoelectrophoresis using yolk antibodies from immunized chickens. J. Immunolog. Methods. 69:1-7; Larsson, A. et al. 1988. Chicken antibodies: a tool to avoid false positive results by rheumatoid factor in latex fixation tests. J. Immunol. Methods. 108:205-208; Larsson, A. et al. 1992. Chicken antibodies: a tool to avoid interference by complement activation in ELISA. J. Immunol. Methods. 156: 79-83; Larsson, et al 1993. Chicken antibodies: taking advantage of evolution. A review Poultry Sci. 72: 1807-1812; Schade, R. et al 1996. The production of avian (Egg yolk) antibodies: IgY. Atla. 24:925-934).
Regarding their therapeutic application, the IgY have been used as immunotherapy in different fields of science, for example, the oral administration of egg yolk immunoglobulins has prevented infections by rotavirus in mouse, bovines and porcins among others (Ikemori, Y. et al. 1992 Protection of neonatal calves against fatal enteric colibacillosis by administration of egg yolk powder from hens immunized with 1<99-pillated enterotoxigenic Escherichia coli. Am. J. Vet. Res. 53:2005-2008; Kuroki, M. et al 1994. Passive protection against bovine rotavirus in calves by specific immunoglobulins from chicken egg yolk. Arch. Virol. 138: 143-148; Marquart, R. 1998. Antibody-loaded eggs for piglets: prevention of baby pigs from diarrhea. Proc. 2nd international Symposium on Egg Nutrition and Newly Emerging Ovo-Technologies. Alberta, Canada).
Even the egg yolk IgY immunoglobulins have been used as antivenins against snakes and scorpions which may be injected for neutralizing the toxins with no risk of the common anaphylactic reactions found in the antivenins made in horse (Larsson, et al 1993. Chicken antibodies: taking advantage of evolution. A review Poultry Sci. 72: 1807-1812). Another application has been to prevent tooth decay in humans caused by Streptococcus mutans (Hatta, H. et al. 1997. Passive immunization Against Dental Plaque Formation in Humans: Effect of a Mouth Rinse containing Egg Yolk Antibodies (IgY) Specific to Streptococcus mutans. Caries. Res. 31:268-274).
In the case of the above mentioned animal diseases, several control and prevention measures have been developed through time due to the extension and economic impact of said diseases. Among the strategies for combating same we find the inactivated vaccines and the live virus vaccines. Nevertheless, none of these strategies has been 100% efficient.
In the specific case of the RNA viruses such as the PRRS virus or the PED virus, the lack of control is largely attributable to its high mutation index. This is a common feature among the RNA viruses arising from the lack of proofreading activity of the RNA polymerase. Thus, this failure along with the fast replication kinetics of the virus increase the risk of mutation and emergence of quasispecies (Manreetpal Singh Brar, Mang Shi, Raymond Kin-Hi Hui and Frederick Chi-Ching (2014) Leung mail Genomic Evolution of Porcine Reproductive and Respiratory Syndrome Virus (PRRSV) Isolates Revealed by Deep Sequencing. PLOS one). It has been reported that the genetic variation of the PRRS virus is between 0.48 and 1.32% (Murtaugh M., (2012) Use and interpretation of sequencing inf PRRSV control programs. Allen D. Leman Swaine Conference. Veterinary Continuing Education). These features of the PRRS virus explain the inappropriate protection obtained by vaccines.
One of the strategies used for preventing the diseases caused by these viruses are the autovaccines, which implies the development of vaccines not only by country, but by regions, on the contrary, the prevention by these means would not suffice.
There has also been observed that in the case of the virus causing PRRS, same is neutralized using immunoglobulins derived from the mammal serum (U.S. Pat. No. 5,489,805). These results teach that the immunoglobulins are an alternative for the treatment of ARN viruses. Nevertheless, the shortcoming with this alternative is that the antibodies thus obtained are unviable.
The application of immunoglobulins obtained from the egg yolk (IgY) has already been used in several applications of animal health and prophylaxis and also in humans. The researches performed by Akita and Nakai (Akita, E., Nakai, S. (2000). Egg nutrition and biotechnology, CAB International, New York, p. 301), show that the protective role of IgY against infectious agents is mainly attributed by its capacity of preventing the colonization of, or neutralizing the toxins.
Therefore, an object of the present invention refers to the obtention of avian derived concentrated IgY immunoglobulins formulations, which are effective and safe against several agents that infect animals, including, for example, the PRRS virus, the Porcine Epidemic Diarrhea (PED) virus, the White Spot Syndrome Baculovirus Complex, bacteria causing bovine mastitis, Actinobacillus pleuropneumoniae and coccidiosis, as well as the intoxications caused by trichothecenes, as its feasibility could directly influence towards an important decrease in the expenses associated with vaccination processes and more importantly, would highly reduce the productive losses associated with said diseases.